This invention relates to agglutination detection and more particularly to a method and apparatus to continuously perform a centrifugal agglutination and detection operation.
Agglutination is the clumping of a suspension of cellular or particulate antigen by a reagent, usually an antibody. Agglutination can occur when an antibody binds to corresponding antigen sites on at least two cells or particles. Large clumps of agglutinated particles, observable by the naked eye, can be formed if the strength of the bonds formed by the antibodies between particles is sufficient to withstand the shear forces tending to break up the agglutinates. Smaller clumps still may be observable with the aid of a microscope or by photodetection means.
The widespread us of agglutination based tests has led to many attempts to automate the procedure. One attempt used in blood banking employs a U-bottomed microplate that allows the simultaneous testing of many individual samples. Typically, erythrocytes are mixed with anti-sera or a plasma sample which might aggregate them. After an appropriate incubation period, the erythrocytes are centrifuged in the microplate and then are resuspended manually or with mechanized agitation. The erythrocytes are allowed to settle and the pattern of cells formed after settling is examined. If the cells are agglutinated, a clump of erythrocytes or "cell button" will settle to the bottom of the U shaped well. Unagglutinated cells will be more uniformly dispersed over the bottom of the well after similar treatment. Differentiation between aggregated and unaggregated erythrocytes can be automated with a photometric detection system that discriminates between the pattern formed by a cell button and that formed by uniformly settled cells.
The foregoing technique has many drawbacks. For example, some agglutination reactions of interest are held together by weak bonds and the agitation may break up the weak agglutination. Thus, there is limited ability to discriminate between no agglutination and weak agglutination. Furthermore, standardizing shear stresses exerted upon the cells during agitation is difficult as these stresses are affected by sample dependent properties which the experimenter cannot control.
Alternative approaches are described in U.S. Pat. No. 4,303,616 and 4,466,740. A microtiter plate having wells with protrusions or depressions on an inclined bottom surface is used to eliminate resuspension and centrifugation steps. As before, erythrocytes and plasma or anti sera are mixed in wells and, after an appropriate incubation period, the reactants simply are allowed to settle onto the inclined bottom surface of the microplate. Essentially, the cells initially captured by the protrusions or depressions act as a base upon which agglutination occurs. When agglutination is present, a greater percentage of cells are captured by the protrusions or depressions, and many fewer cells settle to the bottom of the test vessel. The difference in the pattern produced when there is agglutination as opposed to when there is no agglutination can be read with a photometric detection system.
The failure of the foregoing system to use centrifugal force to pack the erythrocytes has several drawbacks. First, the cells take a long time to settle, thus making this system impractical for many applications. Second, the system lacks the sensitivity achieved when cells are packed together with centrifugal force. Third, the settling patterns created under such a system are extremely delicate and subject to disruption by slight motion. Therefore, in one commercial embodiment of the foregoing system, high cost vibration free transport mechanisms are required for the microplates. Fourth, only positive or negative results are provided and, thus, it is not possible to grade the strength of the agglutination reaction. Finally, the foregoing system does not integrate well with protocols that require washing the cells.
U.S. Pat. No. 4,148,607 discloses an agglutination test that utilizes a vessel having a sloped rear wall upon which reactants are pelleted by centrifugation. After cessation of centrifugation, there is a waiting period during which the fluid the cells were pelleted in is allowed to fall away from the "cell button". The "cell button" is then given the opportunity to move under the influence of gravity along the sloped rear wall. The movement is detected by an optical instrument and correlated to the presence or absence of agglutination.
Since agglutination occurs under the forces of centrifugation, this method does not have all of the same drawbacks as U.S. Pat. Nos. 4,303,616 and 4,466,740. However, the sensitivity and reproducibility of the method is dependent upon the environment which the agglutinated particles, e.g., red blood cells, experience during the settling process. In the previous technique, the flow of the fluid along the sloped wall is dependent on the fluid wall interface and the design of the apparatus allowed fluid to flow away from the aggregated red blood cells, thereby exposing the cells to air during settling and causing the buoyant density of the cells to fluctuate several fold. As a consequence, the sensitivity and reproducibility of the prior method also varied.